Hello, I´ve been trying to transfer a 11 kb plasmid (pCRISPomyces ADDGENE) from E. coli ET12567 to Streptomyces mobaraensis. I have done it three times already with two different methods and I haven´t gotten any exconjugants. Someone has any advices or conjugation assays protocols that could share with me?
Thank you, but yes I have done that protocol already, which is the one from the original article. And still can´t get any excojugants.
Dear
Please check constructive articles about conjugaison.
-An Efficient Intergeneric Conjugation of DNA from Escherichia coli to Mycelia of the Lincomycin-Producer Streptomyces lincolnensis. Lei Du, Rui-Hua Liu, Li Ying, and Guang-Rong Zhao. Int J Mol Sci. 2012; 13(4): 4797–4806.
-Developing an efficient and reproducible conjugation-based gene transfer system for
bifidobacteria Wilfredo Dominguez and Daniel J. O’Sullivan. Microbiology
(2013),159, 328–338.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3344245/
http://www.openwetware.org/wiki/Streptomyces:Protocols/Conjugation
http://www.microbiologyresearch.org/docserver/fulltext/micro/159/2/328_mic061408.pdf?expires=1490982643&id=id&accname=guest&checksum=E30C3266A3F25DC3DC79400BA4C54FE0
https://www.elabprotocols.com/protocols/#!protocol=504
Please check the following articles:
I'm not sure if they will be helpful, and I've been out of the loop for many years, but I am with a group that did these types of conjugations early on and they worked quite nicely. Also I worked on transduction of plasmid DNA with Streptomyces and FP43. That type of DNA transfer was possible using the tra function cloned into PIJ702. I forget what it was called, I cloned the tra function from FP43 into PIJ702 and was able to transfer some DNA cloned into that vector into Streptomyces species quite widely. Here are the conjugation references using the protocols that I had success with: "Molecular Cloning and Physical Mapping of the Daptomycin Gene Cluster from Streptomyces roseosporus", Margaret McHenney et al. and "Complex Enzymes in Microbial Natural Products Research, Part A... https://books.google.com/books?isbn=0080923356, or possibly some references to Patricia J Solenberg "Applications of Transposition Mutagenesis in Antibiotic Producing Streptomyces" Richard H. Baltz et al. There may be modifications to the original protocol in these papers that would help you. I know that Pat fiddled with that protocol before I used it, but I don't know what modifications if any she made, so you may want to look at her work. This paper might be interesting: Characterization of the Replication, Transfer, and Plasmid/Lytic Phage Cycle of the Streptomyces Plasmid-Phage pZL12▿ † Li Zhong,‡ Qiuxiang Cheng,‡ Xinli Tian, Liqian Zhao, and Zhongjun Qin* as well as :
Transduction of plasmid DNA in Streptomyces spp. and related genera by bacteriophage FP43.
McHenney MA1, Baltz RH
Hi, I came across this old thread and just want to share with you that we couldn't get exconjugants from conjugation of E coli ET12567/pUZ8002::pCRISPomyces and Streptomyces albidoflavus J1074 (formerly known as albus J1074)/S. ceolicolor A3(2)/other actinomycetes isolates. Multiple conjugations were repeated for each strain and still no exconjugants. We think that there are some genetic components that are toxic to Streptomyces, therefore we could not get any exconjugants at all.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology